1. Field of the Invention
The present invention relates to a method and apparatus for determining one or more characteristics of particles using multiple fluorescence analysis, and more particularly, concerns a flow cytometry method and apparatus in which multi-color fluorescence analysis may be performed with single wavelength excitation from a light source.
2. Background Description
There are a number of instruments in which the detection of fluorescent characteristics of particles, cells or the like provides valuable information relating thereto. Fluorescence microscopes, flow cytometers and image microscopes, for example, are some of the instruments wherein the detection of fluorescence provides the investigator or user with data concerning the particles or cells under analysis.
In flow cytometry apparatuses, cells or other particles are caused to flow in a liquid flow stream so as to facilitate the investigation of certain characteristics thereof. In general, a flow cytometry apparatus is useful for identifying the presence of certain cells or particles of interest, enumerating those cells or particles and, in some instances, providing a sorting capability so as to be able to collect those cells or particles of interest. In a typical flow cytometry apparatus, a fluid sample containing cells is directed through the apparatus in a rapidly moving liquid stream so that each cell passes serially, and substantially one at a time, through a sensing region. Cell volume may be determined by changes in electrical impedance as each cell passes through the sensing region. Similarly, if an incident beam of light is directed at the sensing region, the passing cells scatter such light as they pass therethrough. This scattered light has served as a function of cell shape and size, index of refraction, opacity, granularity, roughness and the like. Further, fluorescence emitted by labeled cells, or autofluorescent cells, which have been excited as a result of passing through the excitation energy of the incident light beam is detectable for identification of cells having fluorescent properties. After cell analysis is performed by the flow cytometry apparatus, those cells that have been identified as having the desired properties may be sorted if the apparatus has been designed with such capability.
Instruments such as flow cytometry apparatuses are particularly useful for researchers and investigators studying various responses, reactions and functions of the immune system. Immunofluorescence studies as well as fluorescence immunoassays assist the investigator in identifying and targeting select cells of interest so that disease states, conditions and the like may be properly characterized. In addition to immune system investigations, fluorescence analysis is also quite beneficial in cell biology and morphology investigations, including the study of the substructure of cellular material.
In relying upon fluorescence to provide data and information about cells, the mechanics of performing tests for the fluorescence response is a major consideration in the design of the instrument as well as the results to be obtained. Specifically, the fluorescent markers, whether such markers be fluorescent stains or dyes, are typically excited by light energy. Usually there is an optimal wavelength which provides the greatest level of excitation for the fluorochromatic marker being used. Once excited, fluorescence emission occurs typically at wavelengths different from the wavelength of excitation. Fluorescence analysis instruments, whether fluorescence microscopes, image analyzers or flow cytometers, are generally designed to detect the fluorescence emission at the wavelength of emission maxima where the fluorescence signal is strongest.
Fluorescence analysis of cells or particles in which multiple fluorescence emissions may be simultaneously detected is preferred by many investigators because more information about the cells may be gathered. If an arc lamp is employed as the source of excitation light, the spectrum of light available to excite fluorochromatic markers is rich so that different fluorochromes may be excited by a single light source over a wide range of wavelengths. However, the energy level of light energy available for excitation purposes is generally weak so that the resultant emitted signal from the fluorochromatic markers is difficult to detect and process. Other factors, such as the size of the light emitting elements in the lamps, also affect the resultant signals from the fluorochromatic markers. On the other hand, use of a laser for fluorescence excitation purposes provides a significantly higher intensity of the signal, but the light produced by the laser is monochromatic, i.e., at a single wavelength. This has been a limitation for fluorescence analysis investigations since one laser and associated optical and electrical circuitry have been a practical requirement for each fluorochromatic marker to be detected on the cells or particles. In at least one reported instance, Loken et al., "Two-Color Immunofluorescence Using a Fluorescence-Activated Cell Sorter," the Journal of Histochemistry and Cytochemistry, vol. 25, no. 7, pp 899-907, 1977, a single argon-ion laser provided the excitation energy for the simultaneous excitation of fluorescein and rhodamine. Thus, Loken et al. gained the benefit of using one laser excitation source to obtain information about cells associated with two different fluorochromatic agents.
In order to keep the expense and complexity of fluorescence instruments to a minimum while being able to obtain fluorescence emission data from many different fluorescence markets, it would be desirable to be able to use a light source which provides a single wavelength of excitation (at relatively high intensities) for exciting a plurality of fluorescence markers that emit fluorescence at spectrally separated wavelengths, each of which may be individually detected. Although many fluorescent stains and dyes have been available for DNA studies and cell surface marking, it has been unknown to provide a combination of three or more fluorescent markers which are excitable at a single wavelength and produce fluorescence emissions which are sufficiently spectrally separated so that they may be individually detected. As mentioned above, Loken et al. used an argon-ion laser to provide a single wavelength for the excitation of two dyes. Certain new fluorescent conjugates recently have been described in U.S. Pat. Nos. 4,542,104 and 4,520,110. present invention, therefore, is directed to achieving the desired goals set forth above in which a single wavelength of excitation excites at least three fluorescence markers which may be individually detected.